生物化工
应用微生物工程应用微生物工程
    产氢细菌的产氢能力不高是限制生物制氢技术发展的重要因素。运用分子生物学的手段对菌种进行改造,是提高生物产氢效率的重要方法。乙醇和乳酸是产酸克雷伯氏菌Klebsiella oxytoca HP1厌氧发酵产H2的主要副产物，切断无益副产物能提高H2产量和底物转化率。本研究以K. oxytoca HP1作为受体，构建了同源双臂整合载体pTA-Str和pLDH-Tr。利用内源平台双交换原理，将质粒pMHE6上携带的氨基葡萄糖苷腺苷转移酶基因（Aminoglycoside-3'-adenyltransferase，aadA）外源表达盒序列（含T7启动子，aadA 编码框，T4终止子）定点插入到受体菌细胞染色体乙醇脱氢酶系(含乙醛脱氢酶和乙醇脱氢酶活性)基因adhE ＋868～＋869区 (adhE起始编码框为＋1)位点上。并在此基础上以质粒pTA-Str介导K. oxytoca HP1 adhE的插入失活，获得了具有链霉素抗性的adhE－突变株。运用类似方法将pBR322上携带的四环素抗性蛋白基因（tetracycline resistance protein，tet）外源表达盒序列（含P1、P2启动子，tet 编码框）定点插入到受体菌细胞染色体乳酸脱氢酶基因LDHa ＋518～＋519区 ( LDHa 起始编码框为＋1)位点上。在此基础上以质粒pLDH-Tr介导K. oxytoca HP1 LDHa的插入失活，获得了具有四环素抗性的LDHa－突变株。葡萄糖发酵实验结果表明, 相同发酵条件下, adhE－突变菌比野生菌的产氢量提高了16.07%, 乙醇产量下降了70.47%；LDHa－突变株比野生菌的产氢量提高了20.35%。本研究构建的Klebsiella oxytoca HP1突变株adhE－和LDHa－，将为进一步研究高效产氢系统奠定基础。   [英文摘要]：     The low bacterial H2-production ability would be the choke point on the development of bio-hydrogen technology. It is a potential method for enhancing H2-production by using molecular biological technology. Ethanol and lactatic acid are the main byproduct of anaerobic H2-producing fermentation in Klebsiella oxytoca HP1.This can be effectively solved by blocking the pathways leading to the byproducts that are of no benefit to H2-production.  In this study，the homologous integration plasmid vector pTA-Str and pLDH-Tr were constructed using K. oxytoca HP1 as host cell. The adhE genes, which encoded for acetaldehyde dehydrogenase and alcohol dehydrogenase, were cloned into plasmid pMD18-T to generate plasmid pTA. By inserting Aminoglycoside-3'-adenyltransferase (aadA) cassette into the coding region of adhE ＋868～＋869 into plasmid pTA, the homologous integration plasmid vector pTA-Str was constructed. The amplified DNA fragment 5’-adhE-aadA-adhE-3’ from pTA-Str was transformed into K. oxytoca HP1 and the mutant strain adhE－ was sceened. Using the similar strategy, The LDHa genes, which encoded for lactatic acid dehydrogenase, were cloned into plasmid pUC19 to generate plasmid pLDH. By inserting tetracycline resistance protein (tet) cassette into the coding region of LDHa ＋518～＋519 into plasmid pLDH, the homologous integration plasmid vector pLDH-Tr was constructed. Mutant strain LDHa－ was screened after pLDH-Tr was transformed into K. oxytoca HP1. Compared with the H2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant strain adhE－ increased by 16.07% and ethanol concentration decreased by 77.47%, and the hydrogen yield of the mutant strain LDHa－ increased by 20.35%.     In this study, we obtained Klebsiella oxytoca HP1 adhE－ and LDHa－mutant strains. They offered platform for future research on high bacterial hydrogen production.